Microbial communities from the Huaibei Coalfield alter the physicochemical properties of coal in methanogenic bioconversion

Accepted Manuscript Microbial communities from the Huaibei Coalfield alter the physicochemical properties of coal in methanogenic bioconversion

Bobo Wang, Zhisheng Yu, Yiming Zhang, Hongxun Zhang PII: DOI: Reference:

S0166-5162(18)30619-0 https://doi.org/10.1016/j.coal.2018.12.004 COGEL 3133

To appear in:

International Journal of Coal Geology

Received date: Revised date: Accepted date:

6 July 2018 7 December 2018 10 December 2018

Please cite this article as: Bobo Wang, Zhisheng Yu, Yiming Zhang, Hongxun Zhang , Microbial communities from the Huaibei Coalfield alter the physicochemical properties of coal in methanogenic bioconversion. Cogel (2018), https://doi.org/10.1016/ j.coal.2018.12.004

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Microbial communities from the Huaibei Coalfield alter the

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physicochemical properties of coal in methanogenic bioconversion

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Bobo Wanga, Zhisheng Yua,b,* [email protected], Yiming Zhangc, Hongxun Zhanga

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Yuquan Road, Beijing 100049, P.R. China

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Road, Beijing 100085, P. R. China

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Environmental Protection Bureau of Shunyi District, Beijing 101300, China

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Corresponding Author.

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Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing

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College of Resources and Environment, University of Chinese Academy of Sciences, 19 A

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Abstract

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The relationships among the production of methane, the physicochemical properties of coal, and

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the composition of microbial communities are poorly understood in methanogenic bioconversion.

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In this study, we investigated the changes in microbial communities during the methanogenic

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process of coal based on culture-dependent methods as well as the physicochemical properties of

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the coal samples. The process of methane production could be clearly divided into four phases (lag,

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log, peak, and stationary phases). The initial bacterial communities in the cultivation were

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predominantly Bacteroidales, Actionmycetales, and Bacillales; the archaeal community was

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present at values below the detection limit. However, distinct changes in bacterial communities

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were noted at the log phase of methane production. Bacteroides species accounted for more than

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80% of the total bacterial community, and acetotrophic Methanosarcina was the only archaeal

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community. Interestingly, Clostridiales increased considerably during the first 2 weeks, but

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decreased thereafter, indicating that Clostridiales may play a unique role during the initial stage of

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methanogenic coal bioconversion. Furthermore, the final coal sample showed decreased C and O

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contents and increased N and H contents. Volatile and ash contents as well as microporosity were

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also higher than those in the initial state. These results suggested that methanogenic coal

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bioconversion was a complex biochemical process and that the physicochemical properties of coal

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were altered in methanogenic bioconversion. Moreover, these findings may facilitate the

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development of strategies to improve the production of biomethane utilizing coal.

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Keywords: coal; biomethane; microbial community structure; bioconversion; methanogenesis

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1. Introduction

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Coalbed methane (CBM) has become an important non-conventional energy resource worldwide

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(Moore, 2012; Strąpoć et al., 2011). Two mechanisms for the production of CBM have been

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identified, i.e., thermogenic and biogenic processes (Rightmire et al., 1984). Due to its underlying

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environmental and industrial advantages relative to physical and chemical methods of coal

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transformations (Fallgren et al., 2013; Hosseini and Wahid, 2013), coal bioconversion to methane

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may have broad applications in the clean energy field.

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Methane in coal seams is generated in subsurface and deep coal basins by thermogenic,

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geological, or microbial breakdown of complicated coal high molecular polymers (biogenesis)

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(Fakoussa and Hofrichter, 1999; Harris et al., 2008). Many reports have confirmed the biogenic

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origins of CBM in some CBM reservoirs through isotope analysis and suggested that real-time

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methane production occurs in in-situ coal seams (Butland and Moore, 2008; Golding et al., 2013;

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Hamilton et al., 2014; Susilawati et al., 2013). To this end, CBM researchers have focused on how

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to stimulate methane production in situ by promoting the activity of native microbial communities

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to degrade coal. These real-time methane bioreactors have been evaluated in several coal bas ins

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worldwide, such as the Powder River coal basin, Qinshui coal basin, Illinois basin, and Surat basin

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(Green et al., 2008; Guo et al., 2012; Papendick et al., 2011; Zhang et al., 2016b). In addition, a

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number of studies using culture-independent and -dependent methods have revealed that the

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presence of extensive microbial populations can generate methane from coal in the laboratory

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(Guo et al., 2012; McIntosh et al., 2008; Penner et al., 2010; Rathi et al., 2015; Robbins et al.,

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2016; Zhang et al., 2016a). These studies have verified that clean bioenergy methane can be

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obtained via coal bioconversion. Methanogenic coal bioconversion is the consequence of coal and microbial community

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interactions. A detailed analysis of the dynamics of microbial community composition is necessary

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to determine the complicated synergistic or antagonistic effects of these processes in communities

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and to improve the efficiency and stability of biomethane production. Despite improvements in

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our understanding of the characteristics of microbial communities, we still have a very limited

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understanding of the dynamic changes of microbial communities responsible for this process

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(Susilawati et al., 2015; Yang et al., 2018) and little is known about changes in the

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physicochemical properties of coal during methanogenic coal bioconversion by the indigenous

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methanogenic consortia.

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A putative metabolic pathway involved in bioconversion of coal to methane has been

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proposed by Strapoc et al. based on their studies and previous work in other laboratories (Jones et

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al., 2010; Strąpoć et al., 2011). They suggested that coal degradation to methane begins with

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hydrolysis of aromatic, polyaromatic and other hydrocarbon substrates into alcohols, volatile fatty

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acids, and other organic acids. These organic acids are further degraded into methanogenic

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substrates, such as methanol, H2 /CO2 , and acetate. Bacterial species from Clostridium, Pelobacter,

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Bacteroides and Spirochaetes were often detected in the coal seam environment and associated

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production waters, which are involved in fermentation, sulfur metabolism, hydrogen, and

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hydrocarbons hydrolysis (Aklujkar et al., 2012; Sträuber et al., 2012; Wrighton et al., 2012).

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Methanogenic communities are composed of three types of functional metabolic consortia,

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including fermenting, syntrophic and methanogenic species (Iram et al., 2017). The most common

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substrates for methanogenic species in biomethane production are hydrogen/carbon dioxide,

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acetate, and C1 compounds. In terms of these different types of substrates, methanogenic species

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can be classified as hydrogenotrophic, acetoclastic, and methylotrophic (Meslé et al., 2013; Penger

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et al., 2012). In addition, exceptions to these common substrates are different methoxylated

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aromatic compounds, which are substrates for Methermicoccus sp. (Mayumi et al., 2016). These

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various types of methanogenesis suggest that different methanogenic communities induce distinct

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and extensive methanogenic processes.

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In this study, we established an anaerobic bioreactor to perform methanogenic coal

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bioconversion and monitor methane production, and we used this reactor to assess changes in

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bacterial and archaeal community compositions during different phases of methane production.

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In addition, we examined changes in the physicochemical properties of coal. These results

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provide important insights into the optimization of strategies to improve the production of

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biomethane utilizing coal.

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2. Materials and Methods

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2.1 Sampling site

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The Huaibei Coalfield is a primary coal field located in Anhui province, China (Fig. 1).

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Annual production of coal is over 30 million tons. Most coal is used for industrial fuel and by

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power plants (Zheng et al., 2008).The Luling coal mine is located in Huaibei Coalfield with three

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main work seams (seams 8-10). The CBM of Luling coal mine is mixed with secondary biogenic

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gas, and the no. 8 seam has high abundant reserves of coal and methane (Zhou et al., 2014). Thus,

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this seam was the target for analysis of methanogenic coal bioconversion in this study. The

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estimated proportion of thermogenic gas is ranged from 48.54% to 52.15% in the no. 8 coal seam

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of the Luling coal mine, whereas that of biogenic gas ranged from 47.85% to 51.46% (Bao et al.,

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2014).

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Fig. 1. Location of the Huaibei coal mine used for sample collection.

2.2 Sample collection and preparation

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The collected coal samples were obtained from the inside of newly exposed mining faces to

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minimize the oxygenation degree of coal samples. Fresh coal samples were placed in sterilized

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glass bottles and stored at 4 °C in an anaerobic chamber before culture. In the laboratory, the coal

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was ground to coal powder using a mortar and pestle and sieved through a 200-mesh sieve to

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collect fractions containing particles less than 250 μm in diameter in an anaerobic chamber

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(XinmiaoYQX-11; Shanghai, China). The coal powder was separated into two parts, which were

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used for analysis of physicochemical properties and for bioconversion. The coal powder was used

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as the sole energy and carbon substrate. Analysis of the physicochemical properties of the coal

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sample was performed by the Test Center of the China Coal Research Institute in Beijing. A

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continuous drainage system was used to drain formation water from the underground mine. The

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formation water sample used as the inoculum was collected in sterilized polyethylene bottles

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anaerobically. The autoclaved bottles were sealed tightly with butyl rubber stoppers and flushed

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with nitrogen gas as reported previously (Susilawati et al., 2015). In the laboratory, the formation

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water sample (3 L) was filtered through sterile 0.22 μm pore-size hydrophobic membrane filters

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(Whatman Japan KK, Japan). Membranes containing the filtered microbes from formation water

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were used for subsequent culture with coal.

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2.3 Detection of methanogenic coal bioconversion

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To assess changes in microbial communities during the methanogenic coal bioconversion

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process, microorganisms from formation water were used as the inoculum, and crushed coal

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powders were used as the carbon substrate. The basal anaerobic medium contained the following

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(in g/L): MgSO4 ·7H2O (3.45), KCl (0.335), NH4 Cl (0.25), NaCl (11.0), MgCl2 ·2H2O (2.75),

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K2 HPO4 (0.14), and CaCl2 ·2H2 O (0.14). In addition, the medium contained 40 ml of 1.25% Na2 S

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-1.25% cysteine, 1 ml of 0.2% Fe(NH4 )2 (SO4)2, 10 mM HEPES buffer (pH 7.5), and 10 ml/L each

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of vitamin solutions and trace metals. The vitamin solution contained the following (in mg/L):

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pyroxidine HCl (10), folic acid (2), biotin (2), riboflavin (5),thiamine HCl (5), lipobenzoic acid (5),

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vitamin B12 (0.1), nicotinic acid (5), and lipoic acid (5).The trace mineral solution contained the

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following (in mg/L): MnCl2 ·4H2 O (100), ZnCl2 (70), FeCl2 ·4H2 O (1,500), H3 BO3 (36),CuCl2 (2),

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NiCl2 ·6H2 O (24), NaMoO4 (6), CoCl2 ·6H2 O (190), AlK(SO4 )2 (10), and 10 ml/L of 25% HCl.

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Cultures were prepared in 1-L serum bottles (Shuniu Glass), in which 10 g coal powder

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substrate was combined with 350 mL autoclaved basal medium. The bottles were capped with

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butyl rubber stoppers, and the filtered membrane of the formation water as the inoculant was

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transferred into the autoclaved serum bottles (Fig. 2). Culture bottles without coal powders were

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used as negative controls (no-coal controls). The headspace of the serum bottles was removed

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using a vacuum and then flushed with pure N2 three times to remove O2 . Cultures were incubated

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without shaking at 35°C. The rubber hose, stop valve, and syringe were assembled and crossed

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through the butyl rubber stopper to collect 2 mL culture solution every week until methane

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production reached the stationary phase, at which total methane content no longer increased.

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Before collection, cultures in the bottles were shaken at 100 rpm for 1 h using a magnetic stirring

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device to mix the culture solution. The collected cultures were stored at -80°C until used for DNA

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extraction. Cultivations were performed in triplicate. Methane content in the gas collecting bag of

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the bioreactor was detected weekly using a gas chromatograph (Agilent 7890A; Agilent

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Technologies, USA) equipped with a flame ionization detector.

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Fig. 2. Schematic diagram of bioconversion and extraction process. 1: serum bottle, 2: coal, 3: culture solution and

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microflora, 4: rubber plug of the serum bottle, 5: stainless steel conduit, 6: screw cap of serum bottle, 7: aluminum

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foil gas collecting bag, 8: syinger, 9: rubber hose, 10: circulator bath, 11: magnetic stirring device, 12: stop valve,

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13: gas chromatography, 14: high-throughput sequencing.

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2.4 Microbial community analysis

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2.4.1 DNA extraction and amplification

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The archaea and bacteria in the cultures samples were identified using an Illumina

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HiSeq2500. Collected culture samples were a mixture of three cultures from three independent

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experiments. Genomic DNA from the collected cultures supplemented with 0.2% Tween 80 was

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extracted using a FastDNA SPIN kit (Bio101 Systems, USA) according to the manufacturer ’s

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instructions (Guo et al., 2012). The 16S rRNA genes of archaea and bacteria were used for

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microbial community analysis. The universal bacterial primer set BAC-515F/907R (Xiong et al.,

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2012) and archaeal primer set AR-519F/915R (Lane) were used for amplification of 16S rRNA

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genes of bacteria and archaea, respectively.

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2.4.2 Library construction and sequencing

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Purified PCR products were used to construct the DNA library with a TruSeq® DNA

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PCR-Free Sample Preparation Kit according to the manufacturer ’s instructions. Constructed DNA

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libraries were checked using Qubit and qPCR and then sequenced on a HiSeq2500 PE250

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platform.

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2.4.3 Phylogenetic analysis of sequencing data

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Chimeric sequences were processed by quality control with Qiime (V1.7.0) (Caporaso et al.,

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2010). The operational taxonomic units (OTUs) were defined with Uparse software (v7.0.1001,

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http://drive5.com/uparse/) at a 97% cut-off (Edgar, 2013). The representative sequences for each

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OTU were compared with the SSUrRNA sequence database (Quast et al., 2013). The obtained

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bacterial and archaeal 16S rRNA gene sequences were deposited in the NCBI short read archive

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(SRA) database with Bioproject accession number PRJNA474893.

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2.5 Low-pressure N2 gas adsorption (LPGA) isotherms and scanning electron microscopy (SEM)

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LPGA experiments were performed on a QuantachromeAutosorb-6B/3B apparatus to

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evaluate the microporosity of coal samples before and after bioconversion. The coal samples

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(weighing 2 g) for adsorption analysis were outgassed at 120°C for 12 h under high vacuum to

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remove air and free water. N2 adsorption isotherms of samples kept in liquid nitrogen (77.35 K at

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101.3 kPa) were obtained for analysis of relative pressure (P/P 0 ; the gas pressure/the saturated

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vapor pressure) ranging from 0.01 to 0.995. Based on multiple adsorption theories, such as

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Langmuir, Barrett_Joyner_Halenda, Brunauer_Emmett_Teller, Dubinin_Radushkevich, density

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functional theory and Dubinin_Astakhov (Clarkson and Bustin, 1999; Webb and Orr, 1997), pore

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structure parameters were calculated by the computer software. A specific description of these

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techniques and theories was documented by Kakei et al. (Kakei et al., 1990).

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In order to characterize the porosity of coal samples, developed scanning electron microscopy

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(SEM) methodology was adopted (Klaver et al., 2012). Coal samples collected before and after

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bioconversion were observed by field emission-SEM (Quanta 200F). SEM observations were

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conducted at the Electron Microscopy Laboratory of Peking University.

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2.5 Statistical analysis

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Statistical tests based on analysis of variance (ANOVA) with the method of least significant

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differences at the 5% level were used to determine significant differences in the chemical

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composition of coal before and after bioconversion. All statistical analyses were performed with

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SPSS 17.0.

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3. Results

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3.1 Methane production

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Significantly greater methane content (p < 0.001) was produced from continuous cultivations

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of coal powder cultured with inoculum filtered from formation water (test group, 63.8 μmol CH4

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shown). For the entire culturing process, methane production could be clearly divided into four

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phases (lag phase, log phase, peak phase, and stationary phase; Fig. 3) in the test group.

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Specifically, methane production increased slowly until week 3. After week 3, methane production

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increased dramatically and continued until week 5. After week 5, methane production began to

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decrease and was stopped at week 8 when no increased methane production was detected in all

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repeated experimental controls (Fig. 3).

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Fig. 3. Plots of average methane production from coal bio conversion during the 9-week cultivation using coal and

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inoculum filtered from production water. Error bars represent standard deviation for replicates tubes.

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3.2 Changes in the coal physicochemical properties of coal

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When the methane yield no longer increased in methanogenic coal bioconversion, we found

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that abundant amounts of coal substrate remained in the bioreactor. Thus, we then examined

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changes in the physicochemical properties of coal after methanogenic coal biotransformation. As

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shown in Table 1, ultimate analysis revealed that coal sample from the Luling coal mine is

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characterised by high proportions of carbon, and low proportions of hydrogen, nitrogen, sulfur,

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and oxygen. The volatile matter and ash yields were quite high, up to 30.25% and 14.67%. The

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coal macerals were composed of liptinite, inertinite, vitrinite, and mineral matter. Inertinite and

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vitrinite contents in coal samples were quite high, whereas the liptinite contents and mineral

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matter were relatively low. In addition, coal samples from the no. 8 coal seam had a reflectance

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(Ro, max) of 0.82% ± 0.05%. According to the Chinese classification (GB5751-86) (Chen, 2000),

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coal samples from the Luling coal mine in this study w ere classified as bituminous. The coal

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sample after bioconversion had higher volatile matter and ash, contents and hydrogen, and

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nitrogen contents than the origin coal sample, whereas the carbon, oxygen, and fixed carbon

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contents were decreased. The significance of the change of each chemical component was also

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confirmed by the ANOVA (Table 1). The p value for each chemical components of coal was lower

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than 0.05 except sulfur, which implied that all of the tested chemical components, except sulfur,

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underwent significant changes after bioconversion. All ANOVA analysis results are available in

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supplementary material file (Table S1).

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Table 1. ANOVA analysis for chemical components of coals coal samples before and after bioconversion.

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Yield (%)a

Analysis

BEFORE

SSb

df

M Sb

F value

p value

AFTER

Proximate analyses (dry) % M oisture

14.67±0.02

16.04±0.05

2.84

1

2.84

1688.64

2.10*10-6

30.25±0.03

32.58±0.06

8.13

1

8.13

59675.86

1.68*10-9

55.08±0.06

51.38±0.07

20.52

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20.52

14625.32

2.80*10-9

Ash Volatile matter Fixed carbon

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Ultimate analysis vol. (dry ash free)%

81.74±0.

Carbon

79.13±0.09

10.25

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10.25

4664.85

2.75*10-7

13 4.46±0.02

6.95±0.05

9.29

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9.28

10685.16

5.25*10-8

Nitrogen

1.48±0.03

2.96±0.02

3.27

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3.27

7736.12

1.00*10-7

Sulfur

0.12±0.02

0.15±0.02

0.001

1

0.001

5.57

0.0776

Oxygen

12.19±0.04

10.81±0.05

2.83

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2.83

1846.12

1.75*10-6

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Hydrogen

51.7

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Liptinite

44.3

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Inertinite

1.6

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2.4

--

0.82±0.05

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Petrographic analysis vol.%

matter Reflectance

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Classfication a

Each value shown represents the mean±(SD) of three individual experiments.

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b

SS, between-groups sum of squares. M S, between-groups mean squares.

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Low-pressure N2 gas adsorption/desorption analysis was used to evaluate the microporosity

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of coal in this study. The LPGA isotherms for coal samples before and after bioconversion are

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shown in Fig. 4. The remaining coal substrate exhibited higher adsorption, adsorbing most N2 (> 4

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cm3 /g) at the highest pressure (Fig. 4b), whereas the original coal sample absorbed little N2 (< 1

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cm3 /g), indicating minor microporosity. These results demonstrated that there were significant

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differences in coal microporosity before and after bioconversion.

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Fig. 4. LP-N 2GA isotherms for coal samples before (a) and after bioconversion (b).

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Furthermore, SEM was then performed to supplement the results of LPGA isotherms analysis.

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The combination of SEM observations and LPGA could provide clearer insights into the

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microporosity and pore shape of coal (Nie et al., 2015). The surface images of the coal samples

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before and after bioconversion were enlarged 12,000 times; representative images are shown in

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Fig. 5. The remaining coal substrate samples showed a highly porous structure with numerous

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typical slit-shaped pores compared with the original coal samples. Few open pores were observed

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in the original coal samples (Fig. 5a), supporting the smaller hysteresis loops in Fig. 4a. The

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increased numbers of slit-shaped pores supported the increased microporosity of coal after

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bioconversion. The results of SEM observation were quite consistent with the LPGA isotherm

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analysis.

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5.0 μm

5.0 μm

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5.0 μm

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5.0 μm

Fig. 5. Representative SEM images of coal samples before (a) and after (b) bioconversion. The slit-shaped pores are

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indicated by white arrows. The scanning electron microscope was operating at 10 kV.

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Fig. 6 SEM image of microbial attachment to the coal after bioconversion. The rod shaped and spherical cells are

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indicated by red and yellow arrows, respectively. The scanning electron microscope was operating at 10 kV.

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In addition, SEM observations also intuitively showed that a number of microbes adhered to

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the coal matrix after bioconversion (Fig. 6). The agglomeration and adhes ion of two different

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cellular morphotypes were apparent in the SEM images, the most abundant being 2-4 μm rod

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shaped cells. The other morphotype was spherical cells measuring approximately 1 μm. These

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morphotypes were primarily located on or near the cracks of the coal surface.

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3.3 Dynamic analysis of microbial communities

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In order to elucidate dynamic changes in microbial communities during methanogenic coal

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bioconversion, a deep assessment of the microbial communities in the collected culture solution

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every week in the bioreactor was performed by high-throughput sequencing. In total, 1,197,930

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(676,697 + 521,433) qualitative sequences were obtained. The average sequence lengths of

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archaea and bacteria were 371 and 359bp, respectively. Q30 for bacteria and archaea ranged from

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0.96 to 0.99, suggesting high sequencing accuracy. Additional details on the sequencing data are

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shown in the supplementary information (Table S2).

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Methanogenic coal bioconversion successfully enriched the bacterial and archaeal

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communities. The phylogenetic classification of bacteria and archaea and their closest genera from

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all culture points are listed in Table S3. In total, 56 bacterial species and one archaeal species at

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the genus level were observed during methanogenic coal bioconversion. The microbial

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communities responsible for coal bioconversion had high bacterial and low archaeal diversities in

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the bioreactor. Across all cultures and time points, the bacterial sequences were dominated by

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members of the genera Propionibacterium, Bacillus, and Desulfurispora genera, whereas other

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species belonging to the genera Pseudomonas,

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Paenibacillus, and Shewanella were consistently present at low levels (Table S2). For archaeal

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communities, acetoclastic methanogens from the genera of Methanosarcina were dominant in all

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Paludibacter, Desulfovibrio,

Geovibrio,

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cultures (Fig. 7). The dynamics of microbial communities were analysed in terms of methane production phase

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during the 9-week cultivation. After the first week of cultivation, Actinomycetales, Bacteroidales,

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and Bacillales were the most abundant orders, accounting for 55.93%, 14.37%, and 24.14% of

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total bacteria (Fig. 7a), respectively. After the second week, Actinomycetales, Bacteroidales,

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Bacillales, and Clostridiales were the most abundant orders, accounting for 49.19%, 14.3%, 9.7%

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and 25.25% of the total bacteria, respectively. The relative abundance of the total bacteria in the

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third week was quite different from that in the first and second weeks at the order level. Although

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the relative abundance of Actinomycetales, Bacillales and Clostridiales accounted for large

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proportions of the total bacteria, these orders were far more less than that in the second week (Fig.

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7a). The most abundant order was Bacteroidales, accounting for 77.31% of the total bacteria. After

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week 3, the relative abundances of the bacteria changed only slightly. Bacteroidales was dominant

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from week 4 to week 9, whereas orders Actinomycetales and Clostridiales were the main

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components during these weeks (Fig. 7a). The archaeal community abundance at the genus level is

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shown in Fig. 7b. During weeks 1 and 2, the archaeal community was below the limit of detection.

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Subsequently, the acetotrophic Methanosarcina from the order Methanosarcinale was dominant in

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the archaeal community. As culture time increased, the archaeal relative abundance exhibited a

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nearly steady state from week 3 to week 9. These results indicated that regular changes in the

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microbial community structure occurred during methanogenic coal bioconversion.

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Fig. 7. Relative abundance of 16S rRNA gene sequences in the bioreactor from Illumina HiSeq sequencing for

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bacteria (a) and archaea (b). Archaea were not detected in the first 2 weeks. The sample name in each line

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represents the collected culture sample in the bioreactor from week 1 to week 9, respectively.

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4. Discussion

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Many in situ and laboratory-based studies have demonstrated the occurrence of biogenic methane

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and the methane-forming potential of coal bioconversion; however, a limited understanding of the

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microorganisms responsible for this process is documented. This was a comprehensive study

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detailing changes in the structure of the microbial community and the physicochemical properties

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of coal during methanogenic coal bioconversion in a bioreactor with continuous cultivation. In this

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process, simulative bioconversion equipment was combined with a sample extraction device to

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obtain the extraction via in situ culture to avoid disrupting the anaerobic conditions in the

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bioreactor, thereby ensuring the continuity of the bioconversion process. Our analyses of

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microbial communities and changes in physicochemical properties of coal showed that

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methanogenic coal bioconversion is a complex biochemical process.

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During the 9-week cultivation using coal and inoculum filtered from production water, the

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process of methane production passed through four phases. During the first 2 weeks, no methane

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was detected. However, in a study by Rita Susilawati et al., small amounts methane were detected

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in the lag phase (Susilawati et al., 2015). They suggested that the methane was released from

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adsorbed methane in the coal substrate. In this study, the adsorbed methane may have been

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sweeping off by blowing N2 persistently before the reaction started. Although no biomethane was

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produced, coal bioavailability could be accelerated during this phase, providing the available

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substrates for methanogens, consequently resulting in methane production. Following the initial

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lag phase, a typical log phase was also observed for microbial community growth with coal

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(Fuertez et al., 2017; Susilawati et al., 2015; Yang et al., 2018). Significant methane accumulation

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was detected after week 3, and no increases in methane were observed in no coal controls (data

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not shown). The methane production from the test groups increased over time until week 8.

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Although the acetoclastic methanogen Methanosarcina was detected during week 9, the methane

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yield did not increase. Susilawati et al. suggested that the accumulated toxic by-products from

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microbial metabolism during long-term cultivation may influence the capacity of the

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methanogenesis community to transform coal to methane (Susilawati et al., 2015). Significant changes in the physicochemical properties of coal were found during

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methanogenic coal bioconversion. The remaining coal substrate after bioconversion exhibited

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increased volatile matter and ash contents, higher hydrogen and nitrogen contents, and increased

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microporosity, whereas the carbon, oxygen, and fixed carbon contents were decreased. Zhang et al.

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also found that biotreated coal samples had higher volatile contents than untreated coal in a recent

340

study (Zhang et al., 2017). However, they reported that the carbon and oxygen contents of coal

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were increased after bioconversion. Differences in changes in the chemical components could be

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due to the different compositions of coal samples. The increases in N and H contents could be

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attributed to the ammonia of bacterial strains because of the microbial protein interactions with

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coal. Increased microporosity was detected by LPGA, and coal after bioconversion showed higher

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N2 absorption than that before bioconversion. Although it is not easy to view the inner core

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because of its highly compact structure, SEM can be a well-established technique to determine the

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morphology of the coal surface (Nie et al., 2015). The SEM results further supported the LPGA

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experiments. Four types of pore shapes in coal can often be founded under SEM observation.

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Those are cylindrical pores, slit-shaped pores, wedge-shaped pores, and bottle neck pores. Typical

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slit-shaped pores were observed in the coal after bioconversion. The emerging slit-shaped pores

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seem to be intrinsic in the coal. Bioconversion will consume a part of the coal matrix, exposing

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pores from the inner part of the coal and enlarging the sizes of these intrinsic pores, as showed in

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Fig. 5. Thus, the microbial activity may contribute to the increased slit-shaped pores, which may

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facilitate the interaction between coal and microbial communities. However, this hypothesis was

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based on the observed results and how many pores were exposed and to what extend the pore

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volume was enlarged remain unclear. Further studies are needed to quantitatively verify this

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hypothesis to determine changes in the pore structure. In addition, microbial attachment to the coal

358

was observed in the SEM image. Rod shaped and spherical cells were the major microorganisms.

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This close attachment presumably indicates a mechanism to maintain cellular interactions with the

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insoluble carbon utilised by these microorganisms for growth and energy. Similarly, Guo et al.

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also found spherical bacteria and bacillus to be the dominant microorganisms adhered to the coal

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surface and suggested a significant positive correlation between microbial adhesion behaviour and

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coal bioconversion (Guo et al., 2018). Vick et al. found that spherical and rod shaped morphotypes

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were the most abundant cells attached to the coal disk and that microbial biofilm structure became

365

visible over time (Vick et al., 2016). However, there was no obvious biofilm structure observed on

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the coal after 9-week culture in this study. This is probably because powdery coal was not

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conducive to the formation of biofilm.

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The bacterial and archaeal communities were successfully enriched in methanogenic coal

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bioconversion. Changes in bacterial and archaeal communities were analysed during the different

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phases. During the lag phase, the predominant bacterial members of the community were

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Bacteroidales, Actionmycetales, Bacillales, and Clostridiales species. The members of these

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bacteria can ferment and hydrolyse hydrocarbons and aromatic compounds and have been

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identified previously in several studies of coal associated enrichment cultures (Midgley et al.,

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2010; Robbins et al., 2016; Stephen et al., 2014). Thus, these bacteria were likely the primary

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degraders of coal substrates in the bioreactors. Interestingly, Clostridiales showed an obvious

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increase during the first 2 weeks and decreased thereafter. This result suggested that Clostridiales

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may have had a special role during the initial stage of methanogenic coal bioconversion in our

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cultures. Several studies have shown that members of Clostridiales contained a family of

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anaerobic fermentative bacteria, which can degrade aromatic compounds to produce various

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organic acids (Brenner et al., 2005; Lee et al., 2008). Wei et al. (Wei et al., 2014) also found that

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Clostridiales served as a dominant fermentative bacteria involved in coal biogasification. Thus, the

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contribution of this order in this study is probably related to organic matter degradation and to

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organic acid production for syntrophic bacteria, aceticlastic methanogens, or both in the initial

384

phase of methane production. In the log phase, methane production increased promptly and

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reached the maximum methane production rate at around week 5 in all cultures. Moreover, based

386

on the results from pyrosequencing, the genus Methanosarcina was detected in the extract samples

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from week 3 to week 9 and was the predominant component with nearly 100% of the sequence

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reads. Methanosarcina species are the predominant constituents of the archaeal community,

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consistent with other finding showing Methanosarcina accounts for a large proportion after

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enrichment culture with coal (Yang et al., 2018). Methanosarcina is a representative acetotrophic

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methanogen, and the dominance of Methanosarcina throughout the coal bioconversion process

392

strongly suggested that acetotrophic methanogenesis was the main pathway. From week 4 to week

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9, the relative abundances of bacteria changed slightly, suggesting that the community structure

394

became more stable during methane production. In addition, Propionibacterium species were

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always present at high proportions over the culture course in coal containing cultures, suggesting

396

that Propionibacterium species likely metabolized substrates directly from the coal. Strains of

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Propionibacterium were shown to produce propionic acid and acetic acid efficiently from various

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substrates in fermentation (Babuchowski et al., 1993). Because the proportions of the acetoclastic

399

Methanosarcina had increased after the lag phase, it was likely that this organism syntrophically

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consumed the acetate being produced by Propionibacterium, which was involved in acetogens.

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However, further studies are required to identify the specific mechanisms involved in this process.

402

These results suggested that methanogenesis utilized the hydrolysis and fermentation of end

403

products produced by the bacterial components.

PT l

Surat Basin well 1

l

Sydney Basin Well 1

l

k

Bowen Basin

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Lithgow State Coal Mine

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Sydney Basin(south)

CSMB_4034

j

Dechloromonas sp.

Ishikari Basin

CSMB_839

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Fusibacter sp.

i

CSMB_713

Powder River Basin

Dehalobacter sp.

h

CSMB_561

Illinois Basin

Geovibrio sp.

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CSMB_219

g

Thauera sp.

Ordos Basin

CSMB_146

Ordos Basin (east)

Geosporobacter sp.

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CSMB_101

f

Methanosarcina sp.

e

CSMB_85

Damodar Basin

Bacillus sp.

d

CSMB_31

Canadian Sedimentary Basin

Propionibacterium sp.

c

CSMB_20

MA

Pseudomonas sp.

Jingmen-Dangyang Basin

CSMB_8

b

Acinetobacter sp.

Co-occurance in published studies

Cherokee Basin

CSMB number

a

Taxonomy

Fig. 8 The abundant OTUs in methanogenic bioconversion from the Huaibei coalfield with their assigned CSM B

406

numbers at a 98% similarity cut-off and their co-occurrences (indicated by red squares) across a range of published

407

studies. a (Beckmann et al., 2018), b(Kirk et al., 2015), c (Wei et al., 2014), d(Penner et al., 2010), e (Singh et al.,

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2012), f(Guo et al., 2012), g(Tang et al., 2012), h(Strąpoć et al., 2008), I(Green et al., 2008), j (Shimizu et al., 2007),

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k

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(Li et al., 2008), l(Vick et al., 2018).

410

Vick et al. have developed a new and easily accessible Coal Seam Microbiome (CSMB)

411

reference set of OTU sequences from the 16S rRNA gene of coal seam microbial communities

412

provided in many published manuscripts (Vick et al., 2018). By mapping the 16S rRNA gene from

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the Huaibei coalfield in this study to the CSMB reference set, some of the abundant OTUs

414

identified in methanogenic bioconversion were also found have occurred in other coal basins

415

around the world (Fig. 8). Interestingly, both the dominate OTUs matched in the CSMB reference

416

set were not observed in the Canadian sedimentary basin and Damodar basin. Of the co-observed

417

microbes, the uniquely identified archaea Methanosarcina sp. (CSMB_101) from the Huaibei

418

coalfield were ubiquitous and have been found previously in the majority of coal basin

419

environments. Similarly, the proteobacterial bacteria Pseudomonas sp. (CSMB_20), a commonly

420

observed genus has also been observed in the Surat basin, Sydney basin, Bowen basin, and

421

Ishikari basin. Pseudomonas sp. is known to degrade hydrocarbon, alkane, naphthalene, and

422

polyaromatics (Prabhu and Phale, 2003; Ross and Gulliver, 2016). Members of Acinetobacter sp.

423

(CSMB_8) observed in Surat basin, Sydney basin, Bowen basin and Ordos basin have

424

hydrocarbon degradation capacities (Yousaf et al., 2011). The Rhodocyclales order members

425

Thauera sp. (CSMB_219), observed in Surat basin, Bowen basin, and Sydney basin have been

426

reported to denitrify under anaerobic aromatic compound degradation (Shinoda et al., 2004). The

427

proteobacterial bacteria Dechloromonas sp. (CSMB_4034) only co-occurred in the Sydney basin

428

well 1# across the CSMB reference set is involved in degrading aromatic hydrocarbons

429

anaerobically (Chakraborty et al., 2005; Fry et al., 2009), such as benzene, a likely intermediate of

430

coal depolymerisation (Zheng et al., 2017). However, some dominate OTUs were not mapped to

431

the CSMB reference set. For example, Paludibacter sp. belonging to the Bacteroidales order could

432

not map to any members of the CSMB reference set. This uncommon phenomenon indicated that

433

a subset of microorganisms surviving in the Huaibei coalfield may have limited distributions.

434

5. Conclusion

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This study systematically showed that the bacterial community structure changed over time during

436

methanogenic coal bioconversion and that acetotrophic methanogens contributed to the maximum

437

biomethane production. Notably, the microbial communities changed obviously during the log

438

phase. What is more, the coal samples after bioconversion showed decreased C and O contents

439

and increased N and H contents, volatile matter, ash contents, and increased microporosity. These

440

findings suggested there are complicated interactions among the production of methane, the

441

physicochemical properties of coal, and the composition of microbial communities. Further

442

studies are required to improve the performance of the main groups of microorganisms involved in

443

order to increase process stability and enhance biogas production.

444

Acknowledgments

445

This work is supported by National Natural Science Foundation of China (20877098) and the

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Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB15010200).

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Methane production was divided into lag, log, peak, and stationary phases.

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Significant changes in bacterial communities were observed during the log phases.

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Clostridiales increased obviously during the lag phase, but decreased thereafter.

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Final coal samples showed changed chemical contents and increased microporosity.

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